Acute renal ischemia is a common clinical occurrence associated with considerable morbidity and mortality. The clinical diagnosis known as acute tubular necrosis (ATN) is associated with decreased urine output and a progressive increase in the serum creatinine. During the recovery from ATN, polyuria defined as a urine output greater than 2 liters per day is observed for three to five days. Pathological features of ATN have been associated with alterations in the actin cytoskeleton and a loss of membrane polarity. The polyuric phase of ATN has been suggested to be causally related to the apical location of functional Na, K-ATPase. Once the normal membrane distribution of Na, K-ATPase is reestablished the polyuric phase of ATN resolves. However the prolonged polyuric phase of ATN cannot be reconciled with the best available data estimating rates of membrane turnover in renal epithelial cells. This suggests that newly synthesized Na, K-ATPase may be mis-sorted or targeted to the wrong plasma membrane domain in renal epithelial cells recovering from ischemic injury. The experiments outlined in this proposal, are designed to test the hypothesis that ischemic injury or ATP depletion, disrupts the sorting and targeting machinery of epithelial cells. Specifically, ischemic injury causes cargo (integral membrane) proteins to escape from their specific transport complex. A complementary possibility is that a randomized distribution of t-snares and/or the sec6/sec8 basolateral docking complex is a sequelae of ATP deprivation. This would lead to significant mis-sorting of proteins during recovery from injury. This hypothesis will be tested by examining the protein sorting characteristics of a variety of apical and basolateral membrane proteins and glycolipids during the recovery from ATP depletion. The sorting and transport of proteins will be examine din vivo using low level light microscopy, fluorescent tagged proteins, image processing and biochemical isolation techniques. The subcellular distribution of the sec6/sec8 docking complex and syntaxin isoforms will be studied by immunohistochemical methods and advanced imaging techniques.